Betaine plays an imporant role in plant responses to various environmental stresses.Phosphoethanolamine N-methyltransferas(PEAMT) is the key regulatory enzyme in the biosynthesis pathway of the precursor choline.In this study,a full-lengh cDNA of PEAMT gene(named LbPEAMT) was firstly isolated and characterized from the leaves of L.barbarum.Full length LbPEAMT was 1 808 bp,containing a 1 497 bp ORF and encoding 498 amino acids.Comparative and bioinformatics analyses revealed that the amino acid sequence encoded by this gene(LbPEAMT) was highly homologous(93%) to PEAMTs from other plant species(i.e.,tomato).The predicted second structure of this LbPEAMT protein was composed of 44.98% of alpha-helix,17.67% of extended chain,6.63% of beta-turn and 30.70% of irregular curl.LbPEAMT was a hydrophilic protein containing two of the S-adenosylmethion-ine-dependent catalyzing domains,which were located in the N-terminal of 65-164 aa and C-terminal of 290-392 aa,respectively.Each catalyzing domain contained four motifs named as I,post I,post II,and post III.However,the two catalyzing domains of LbPEAMT were relatively conservative in the structure of protein methyltransferase,phospholipid transferase and methyltransferase of small molecules.

Abscisic acid(ABA)regulates the essential physiological and developmental processes of plants and plays imporant roles in plant responses to various environmental stresses.9-cis-epoxycarotenoid dioxygenase(NCED)is the key regulatory enzyme in the biosynthesis pathway of ABA in higher plants.In the study,a full-lengh cDNA of NCED gene(LbNCED)was fristly isolated and characterized from the leaves of L.barbarum.LbNCED was 2316 bp,containing a 1824 bp ORF and encoding 607 amino acids.Comparative and bioinformatics analysis revealed that the homology amino acid sequence of Lycopersicon esculentum and Solanum tuberosum LbNCED was 90%.At the N-terminus of the LbNCED located a 15 amino acids putative chloroplast transit peptide.Southern blot analysis revealed that it was a low-copy gene in the genome of L.barbarum.Real-time Quantitative PCR(RT-QPCR)analysis showed that LbNCED mRNA most abundantly accumulated in leaves.The RT-QPCR analysis revealed that dehydration and salt stress signficantly enhanced LbNCED transcript expression and ABA content accumulation.

In this study,a full-length cDNA of allene oxide synthase(AOS) gene(named as CrAOS,JQ364955) was cloned from Catharanthus roseus.The gene was 2 118 bp in size containing an open reading frame(1 638 bp) encoding 545 amino acids.Comparative and bioinformatic analysis revealed that the deduced protein of CrAOS was highly homologous to AOSs from other plant species.Southern blot analysis revealed that it was a low-copy gene.Real-time Quantitative PCR(qRT-PCR) analysis showed that CrAOS mRNA accumulated most abundantly in old leaves and least in young alabastrums.The qRT-PCR analysis revealed that wound,low temperature,methyl jasmonic acid,ethylene treatments significantly enhanced CrAOS transcript expression,and salicylic acid had no influence.

In order to clarify the dynamic change of three alkaloids(TIAs including vindoline,catharanthine and vinblastine) and the expression of the associated biosynthetic genes in the Catharathus Roseus leaves(young,medium and old) at different growing stages,high performance liquid chromatography was applied to determine the contents of TIAs at the leaf period,pre-floweringperiod,bud stage,flowering period,fully-mature period 1 and fully-mature period 2 in five pairs of leaves from Catharathus Roseus.In addition,the expression of the associated biosynthetic genes was analyzed by molecular biological technique(real time PCR,FQ-PCT).The results showed that highest contents of vindoline and catharanthine were presented in young leaves while the content of vinblastine was highest in old leaves.The best time for collecting the leaves of Catharathus Roseus was between the stage of alabastrum and flowering since the maximum contents of TIAs could be extracted in this period.The data from the FQ-PCR determination indicated that the expression of TIAs contents in C.Roseus leaves was more affected by the six genes including GGPP,G10H,SLS,STR,D4H and DAT while two genes(ASA,TDC) showed less influence on the production TIAs.